Project Summary/Abstract The broad, long-term objective of this project is to continue the development of new bioprobes and methodologies for analysis of individual cells by flow and image cytometry. These assays will find utility in studies of DNA damage, DNA repair, carcinogenesis/mutagenesis, apoptosis, and in identifying the mechanism of action of antitumor drugs. The first approach analyzes changes in the maximal pixel intensity of fluorescence of DNA-bound fluorescent ligands, a reporter of chromatin relaxation, the initial event in a cell's DNA damage response (DDR). Other probes to be developed utilize phospho-specific antibodies and multiparameter cytometry to measure phosphorylation/activation of key DDRs: histone H2AX, ATM, Chk2 and p53. The probes ability to measure DDR in response to different types of DNA damage will be assessed including that caused by drugs, mutagens and radiation, and correlated with cell cycle phase and induction of apoptosis. The capability of image cytometry to quantify the frequency of individual nuclear foci each representing a DNA double-strand break (DSB) occurring during DDR will be explored. We will also develop an integrated, multiplexed methodology that combines the detection of DDR (induction of 3H2AX, or ATM, Chk2 and p53 phosphorylation) with the measurement of actual DNA damage, assessed by single-cell DNA electrophoresis (comet assay). The image-merge capability of the laser scanning cytometry will be used to integrate the analysis of DDR with the assay of DNA damage in the same individual cells. Another multiplexed assay to be developed will combine the analysis of DDR with DNA repair, measured as unscheduled DNA synthesis (UDS). The incorporation of 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'deoxyuridine (EdU), the latter followed by the cyclo-addition reaction (click chemistry) will be explored and developed so that UDS and DDR can be assessed in the same cell. Using the approaches described above, the potential DNA damage and DDR induced by supravital probes commonly used in cytometry (Hoechst 33342, DRAQ5, DyeCycle Violet and SYTO 17) will be assessed. Particular attention will be given to explore whether DNA damage occurs under conditions in which these probes are used to isolate stem cells as side populations. The detection of DNA damage, especially DSBs, in cytometrically isolated stem cells will be predictive of the carcinogenic/mutagenic potential of the isolation procedure. Evaluation of the role of the tumor suppressor p53 in DDR caused by different genotoxic agents, including antitumor drugs targeting DNA, is another project in this application. The assays we propose to develop will find wide application in many diverse fields including pharmacology, mutagenesis, carcinogenesis, toxicology, experimental and clinical oncology as well as in cell and molecular biology.